A strain of black fungus and its application

Abstract

This invention relates to the field of edible fungi technology, and discloses a new strain of Auricularia heimuer and its applications. The Auricularia heimuer strain JH-Y6 of this invention was deposited at the China Center for Type Culture Collection (CCTCC) on November 20, 2023, with accession number CCTCC No: M 20232280. Auricularia heimuer JH-Y6 possesses high tyrosinase activity, high melanin production, and excellent agronomic traits, and can be used in the screening and breeding of Auricularia heimu, the extraction process of melanin from Auricularia heimu, and its cultivation. It can also be used in the deep processing of food or health products.

Description

Technical Field

[0001] This invention relates to the field of edible fungi technology, and in particular to a new black fungus strain JH-Y6 and its applications. Background Technology

[0002] Black fungus has a crisp, smooth, and refreshing texture, and is rich in various functional nutrients such as polysaccharides, melanin, polyphenols, and flavonoids. It is an edible fungus that is both nutritious and has pharmacological effects. China is the main producing area of ​​black fungus, accounting for more than 99% of the world's total production. The cultivation of black fungus can utilize a large amount of agricultural waste such as sawdust, straw, bran, and cottonseed hulls, realizing the resource utilization of agricultural waste. It is a sunrise industry for promoting circular agriculture and realizing the resource utilization of waste.

[0003] Black fungus is famous for its black color, which is also one of its important commercial characteristics. The ability of black fungus to produce melanin is the main factor affecting the color of its fruiting body. Melanin has functions such as anti-oxidation, anti-radiation, anti-aging, cardiovascular protection, and immune regulation, and has broad application prospects in the fields of medicine, functional foods, and cosmetics. Compared with synthetic pigments, black fungus melanin has advantages such as being green, safe, and healthy, and can serve as a new source of high-quality natural pigments. The melanin synthesis pathway mainly involves the oxidation of the precursor L-tyrosinase, indicating that tyrosinase activity is closely related to melanin synthesis.

[0004] Mutagenesis breeding, compared to traditional hybridization breeding, has advantages such as a shorter breeding cycle and richer genetic diversity, and has already been applied to the selection of new edible fungi varieties. Currently, the most commonly used mutagenesis methods in edible fungi abroad include ultraviolet (UV), X-ray, and 60Co-γ-ray. Each mutagenesis method has its own unique mutation characteristics. Among them, UV mutagenesis is the simplest and most inexpensive method, mainly causing the formation of thymine dimers or cytosine dimers between adjacent pyrimidine nucleotides, unlike ionizing radiation (such as X-rays) which causes changes in DNA bases and DNA strand breaks. Atmospheric pressure room temperature plasma (ARTP) mutagenesis, compared to other traditional mutagenesis techniques, is simple to operate, has mild conditions, high safety, and rapid mutagenesis. It can greatly improve the intensity of strain mutations and the capacity of the mutation library. Combined with screening pressure and high-throughput screening technology, it holds promise as a new method for efficient evolutionary breeding.

[0005] This study aims to breed new strains of black fungus that produce high levels of melanin and exhibit excellent agronomic traits through various mutagenesis methods. This is of great significance for promoting the healthy development of the black fungus industry, the development of functional products, and rural revitalization. Summary of the Invention

[0006] The purpose of this invention is to provide a black fungus strain JH-Y6, which has the characteristics of high tyrosinase activity, high melanin production and excellent agronomic traits.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0008] This invention provides a strain of black fungus, namely black fungus (Auricularia heimuer) JH-Y6, with the preservation number CCTCC No: M 20232280.

[0009] The black fungus JH-Y6 strain of this invention exhibits high tyrosinase activity and high melanin production. The tyrosinase activity of JH-Y6 is on average 2.84 times higher than that of its parent strain, and the melanin content is on average 2.42 times higher. Compared to another mutant strain, Y9, the tyrosinase activity is on average 79.66% higher, and the melanin content is on average 42.38% higher. The ITS sequence of the black fungus strain is shown in SEQ ID NO.1.

[0010] In this invention, the black fungus JH-Y6 was obtained by screening the spore suspension of the parent strain 'Jiyou No. 1' through ARTP-UV combined mutagenesis, that is, ARTP mutagenesis for 20s followed by UV mutagenesis for 100s.

[0011] This invention also includes the following substances produced by the above-mentioned black fungus strain:

[0012] (1) Basidiospores;

[0013] (2) Mycelium;

[0014] (3) Sub-entity.

[0015] The present invention also includes the fermentation broth of the above-mentioned black fungus strain.

[0016] Another objective of this invention is to provide the application of the above-mentioned black fungus strains and their produced basidiospores, mycelia or fruiting bodies in the screening, breeding and cultivation of black fungus.

[0017] Another object of the present invention is to provide the above-mentioned black fungus strain and the basidiospores, mycelia or fruiting bodies produced therefrom, as well as the fermentation broth produced by fermentation, for the extraction of tyrosinase or black fungus melanin.

[0018] Another objective of this invention includes the application of the aforementioned black fungus strain and its basidiospores, mycelia or fruiting bodies, as well as the fermentation broth produced during fermentation, in food or health products.

[0019] The beneficial effects of this invention are:

[0020] This invention utilizes ARTP-UV combined mutagenesis on the spore suspension of the parent black fungus strain 'Jiyou No. 1'. The presence or absence of melanin deposition in the colonies is used for initial screening of the isolated and purified mutant black fungus strains. Secondary screening at the mycelial stage is conducted through clamp observation of hyphae, tyrosinase activity measurement, and melanin content determination. The strains isolated and purified from tissue are then subjected to fruiting body testing to screen the black fungus mutant strains at the fruiting body stage. Affinity testing yielded a new black fungus strain, JH-Y6, which exhibits increased tyrosinase activity and melanin content compared to the parent strain and another mutant strain, Y9. It also demonstrates excellent agronomic traits, with thicker fruiting bodies and higher fresh weight per fruiting body.

[0021] Biological Preservation Information

[0022] The black fungus (Auricularia heimuer) JH-Y6 of this invention was deposited on November 20, 2023, at the China Center for Type Culture Collection, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCCNo: M 20232280. Attached Figure Description

[0023] Figure 1 Colony morphology of mutant black fungus strains JH-Y6 and Y9 and their parent strain 'Jiyou No. 1'.

[0024] Figure 2 Pigment production of mutant black fungus strains JH-Y6 and Y9 and their parent strain 'Jiyou No. 1'.

[0025] Figure 3 Pigment production of mutant black fungus strains JH-Y6 and Y9 and their parent strain 'Jiyou No. 1' after 10 generations of colony culture.

[0026] Figure 4 Observation of clamp linkage of mutagenic black fungus strain JH-Y6 mycelium.

[0027] Figure 5 Tyrosinase activity and melanin content of mutant black fungus strains JH-Y6 and Y9 and their parent strain 'Jiyou No. 1'.

[0028] Figure 6 The fruiting body morphology of the mutant black fungus strain JH-Y6 and its parent strain 'Jiyou No. 1'.

[0029] Figure 7 Affinity of mutant black fungus strain JH-Y6 with its parent strain 'Jiyou No. 1'.

[0030] Figure 8 Phylogenetic tree constructed based on the ITS sequences of mutant black fungus strain JH-Y6 and 24 other mutant strains and the parent strain 'Jiyou No. 1'. Detailed Implementation

[0031] This invention uses the cultivated black fungus strain 'Jiyou No. 1' preserved by the Edible Fungi Research Laboratory of the Institute of Biology, Hebei Academy of Sciences as the parent, and employs ARTP-UV combined mutagenesis technology for mutation breeding to screen and obtain a new black fungus strain JH-Y6 with high tyrosinase activity, high melanin production, and excellent agronomic traits. The new black fungus strain JH-Y6 was deposited at the China Center for Type Culture Collection on November 20, 2023, with accession number CCTCC No: M 20232280.

[0032] This invention employs ARTP-UV combined mutagenesis to mutate the spore suspension of 'Jiyou No. 1'. After the mutagenic spores grow to the point of multispore hybridization, the isolated and purified mutagenic black fungus strains are initially screened based on the presence or absence of melanin deposition in the colonies, and their genetic stability is determined. Secondary screening at the mycelial stage is conducted through clamp observation of hyphae, tyrosinase activity measurement, and melanin content determination. The strains isolated and purified from tissue are then subjected to fruiting body testing to screen the black fungus mutagenic strains at the fruiting body stage. Combining affinity tests and ITS sequence analysis, the black fungus mutagenic strains were identified, and a new black fungus strain with high tyrosinase activity, high melanin production, and excellent agronomic traits was named JH-Y6.

[0033] By measuring the tyrosinase activity and melanin content of mycelial solid-state fermentation, it was found that the tyrosinase activity of JH-Y6 was 2.84 times higher than that of the parent strain on average, and the melanin content was 2.42 times higher than that of the parent strain on average; compared with another mutant strain Y9, the tyrosinase activity was 79.66% higher on average, and the melanin content was 42.38% higher on average.

[0034] The fruiting test and growth index determination of the black fungus mutant strain JH-Y6 and its parent strain showed that JH-Y6 significantly improved the fruiting thickness and fresh weight of a single fruiting body compared with the parent strain; the brightness L... * The range from 0 to 100 represents black to white, with JH-Y6 having a brightness level L compared to its parent. * The agronomic traits are significantly reduced, and the fruiting bodies are darker than the parents.

[0035] Affinity tests between the mutant strain JH-Y6 and its parent strain 'Jiyou No. 1' revealed that JH-Y6 exhibited a raised hyphae pattern, indicating incompatibility. Phylogenetic trees and genetic distances constructed based on the ITS sequences of the mutant strain confirmed that JH-Y6 is a novel strain distinct from its parent.

[0036] The novel black fungus strain JH-Y6, obtained through screening in this invention, possesses excellent characteristics and can be used in the fermentation extraction of tyrosinase and melanin, as well as in the screening, breeding, and cultivation of black fungus. This includes, but is not limited to, the basidiospores, mycelium, and fruiting bodies of strain JH-Y6, and the fermentation broth obtained from the fermentation process. Those skilled in the art can further process the screened novel black fungus strain JH-Y6, its produced basidiospores, mycelium, and fruiting bodies, and its fermentation broth, with or without the addition of other food ingredients or medicinal materials, to produce food or health products.

[0037] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] The parent strain of black fungus used in the following examples, 'Jiyou No. 1', is a black fungus cultivar preserved by the Edible Fungi Research Laboratory of the Institute of Biology, Hebei Academy of Sciences. It has the advantages of fast growth rate in the mycelial stage and strong resistance to Trichoderma, but in the fruiting body stage, the fruiting lobes are thin and the color is light.

[0039] Example 1

[0040] Collection of parent black fungus spores

[0041] Fresh black fungus 'Jiyou No. 1' with thick, intact ear pieces and good growth condition was selected. Under aseptic conditions, the ear pieces were soaked in 75% ethanol for 1 minute, rinsed three times with sterile water, and dried with sterile filter paper. The sterilized ear pieces were then placed in sterile empty petri dishes. After 3 days, the black fungus ear pieces were removed and diluted with sterile physiological saline to an initial concentration of 10. 7 -10 8 A suspension of 'Jiyou No. 1' spores per mL.

[0042] Black fungus ARTP-UV co-mutation

[0043] Following the ARTP-M operating procedure, the prepared 'Jiyou No. 1' spore suspension (initial concentration 10) was... 7 -10 8 ARTP mutagenesis was performed using helium as the working gas, with a treatment distance of 2 mm, a treatment power of 100 W, a gas flow rate of 10 SLM, and a treatment time of 20 s.

[0044] Take 300 μL of ARTP-mutated spore suspension and spread it on a PDA culture dish. Use a UV mutagenesis chamber to mutate the ARTP-mutated spore suspension. The UV power is 11W, the lamp distance is 20cm, and the treatment time is 100s.

[0045] Cultured at 25℃ for 10 days, spore germination was observed. After the induced spores grew to the point where multispore hybridization was carried out, the hybrid colonies were picked and inoculated onto PDA medium.

[0046] Example 2

[0047] Screening of mutant strains of black fungus

[0048] The mutant black fungus strains isolated and purified in Example 1 were inoculated into PDA medium and cultured at 25°C for 15 days. Colony morphology and melanin deposition were observed, yielding 25 mutant strains with melanin deposition. Among them, JH-Y6 and Y9 showed regular mycelial growth. Figure 1 Compared to its parent 'Jiyou No. 1', melanin deposition was significantly higher. Figure 2 JH-Y6, after 10 generations of subculture, still exhibited significant melanin deposition, confirming its stable melanin-producing capacity. Figure 3 ).

[0049] The clamping connections of the mutagenic black fungus strain JH-Y6 were observed under a 40x microscope using the squash method. Figure 4 The mutant black fungus strain JH-Y6 shown has clamp connections and is a binucleate strain, which can be used for black fungus cultivation.

[0050] Example 3

[0051] Determination of JH-Y6 tyrosinase activity and melanin content in black fungus

[0052] Solid fermentation medium: 62.5g wheat bran was placed in a water bath at 60℃ for 5 hours, boiled for 30 minutes, filtered to obtain wheat bran water, 15.0g agar, 1.6g tyrosine powder, and 0.1g copper sulfate were added, and the volume was adjusted to 1L.

[0053] Extraction reagents: pH 6.0 phosphate buffer, 1.5 mol / L sodium hydroxide, 6 mol / L hydrochloric acid, tyrosinase extraction kit (Beijing Solarbio Science & Technology Co., Ltd. BC4050).

[0054] Using a 5mm punch, make holes at the edge of the colony of the mutant strain, inoculate at the center of the PDA medium, and incubate at 25℃ for 7 days. Then, use a 5mm punch to transfer the mycelium to the center of the solid fermentation medium and incubate statically at 25℃ for 15 days. After incubation, starting from the inoculation point, punch five mycelial blocks along the radius of the petri dish to the edge of the dish using a 5mm diameter punch, and place them in 2mL centrifuge tubes for tyrosinase activity assay. Select the remaining mycelial blocks from the same petri dish for melanin content assay. Each treatment was performed in triplicate.

[0055] Tyrosinase activity assay: The sample stored in a 2 mL centrifuge tube was ground using a grinder, and 600 μL of phosphate buffer was added and mixed by inverting. The mixture was centrifuged at 12,000 rpm for 10 min at 4 °C, and the supernatant was collected to obtain the crude enzyme solution. The OD value of the crude enzyme solution after reacting at 475 nm for 9 min was measured using a tyrosinase extraction kit, and the tyrosinase activity was calculated. The amount of enzyme that catalyzes the production of 1 nmol of dopachrome per minute per mL of crude enzyme solution is defined as one unit of enzyme activity.

[0056] Tyrosinase activity (U / mL) = OD ÷ (ε × d) × Vtotal × 102 9 ÷V-shaped ÷T,

[0057] Where Vreaction total: total reaction volume (10) -3 L); ε: molar extinction coefficient of dopachrome (3.7 × 10⁻⁶). 4 L / mol / cm); d: optical path length of the 96-well plate (6.4 mm); 10 9 Unit conversion factor (1 mol = 10) 9 (nmol); V sample: the volume of sample added (0.1 mL); T is the reaction time (9 min).

[0058] Preparation of the melanin standard curve: Weigh 0.005 g of melanin standard and dissolve it in 10 mL of 1 mol / L NaOH solution to prepare a melanin standard with a concentration of 500 mg / L. Take 800, 600, 400, 200, 100, and 0 μL of the 500 mg / L melanin standard, respectively, and add 200, 400, 600, 800, 900, and 1000 μL of 1 mol / L NaOH solution to prepare melanin standards with concentrations of 400, 300, 200, 100, 50, and 0 mg / L, respectively. Measure the OD value at a wavelength of 550 nm. Plot the standard curve using GraphpadPrism 9 software: y = 0.004990x + 0.007478R 2 =0.9999 (y: OD value of the sample at 550nm; x: melanin concentration mg / mL).

[0059] Melanin content determination: The remaining sample was ground in a mortar and pestle and transferred to a 50 mL centrifuge tube. 1.5 mol / L sodium hydroxide was added to bring the volume to 35 mL. The mixture was shaken and sonicated at 250 W for 40 min. After centrifugation at 12000 rpm for 15 min, 5 mL of the supernatant was transferred to a 15 mL centrifuge tube. An equal volume of 6 mol / L hydrochloric acid was added, and the mixture was kept at 80℃ for 10 h for acid precipitation. After acid precipitation, the mixture was centrifuged at 12000 rpm for 15 min, the supernatant was discarded, and 5 mL of 1.5 mol / L sodium hydroxide was added for alkaline dissolution. The OD value at 550 nm was measured, and the melanin content was calculated based on the melanin pure product standard curve.

[0060] Table 1. Tyrosinase activity and melanin content of *Auricularia auricula-judae* strains

[0061] Measurement indicators JH-Y6 Y9 Jiyou No. 1 Melanin (mg / L) 604.36±52.80 424.46±11.24 176.63±52.91 Tyrosinase (U / mL) 43.46±0.49 24.19±2.02 11.31±1.27

[0062] As shown in Table 1 and Figure 5 As shown, the tyrosinase activity of JH-Y6 was 2.84 times higher than that of the parent strain on average, and the melanin content was 2.42 times higher than that of the parent strain on average; compared with another mutant strain Y9, the tyrosinase activity was 79.66% higher on average, and the melanin content was 42.38% higher on average.

[0063] Example 4

[0064] Characteristics of the fruiting body stage of the black fungus mutant strain JH-Y6

[0065] Fresh wood ear mushrooms with thick, intact ear pieces and good growth were selected. Under aseptic conditions, they were soaked in 75% ethanol for 1 minute, rinsed three times with sterile water, and the surface moisture was absorbed with sterile filter paper. The ear flesh was then picked up with sterile forceps in a laminar flow hood, dipped in sodium penicillin, and cultured in PDA medium containing gentamicin for 3-5 days. Healthy hyphae around the tissue block were picked and cultured on normal PDA for 15 days to obtain the isolated and purified mutant strain JH-Y6.

[0066] The following cultivation substrate formula was used: 50% sawdust, 30% cottonseed hulls, 18% wheat bran, 1% gypsum, and 1% sucrose. Fruiting experiments were conducted, with 50 bags of each mutant black fungus strain inoculated. The parent strain 'Jiyou No. 1' was used as a control. The characteristics of the fruiting body stage were investigated through the fruiting experiment.

[0067] The morphology of the fruiting bodies of the black fungus mutant strain JH-Y6 and its parent strain 'Jiyou No. 1' is as follows: Figure 6 As shown in Table 2, the results of the fruiting test and growth index determination of the black fungus mutant strain JH-Y6 and its parent strain are as follows: the fruiting body thickness and fresh weight of a single fruiting body of JH-Y6 are significantly improved compared with the parent strain; the brightness L * The range from 0 to 100 represents black to white, with JH-Y6 having a brightness level L compared to its parent. * The significant reduction indicates that its fruiting body is darker than that of its parent.

[0068] Table 2. Determination of ear-growing indicators of black fungus JH-Y6 and its parent lines.

[0069]

[0070] Example 5

[0071] Identification of the black fungus mutant strain JH-Y6

[0072] 1 Affinity Test between Mutant Strains and Parents of Auricularia heimuer

[0073] Use a 5-mm puncher to punch holes along the edge of the Auricularia heimuer colony. In each petri dish, inoculate the mycelial blocks of the parental strain and the mutant strain at symmetrical positions. Culture at 25 °C for 10 d, and observe and record the mycelial growth and antagonistic situation. The antagonism between colonies can be manifested as gully type, uplift type, color line type, and isolation type. The antagonistic test between the mutant strain JH-Y6 of Auricularia heimuer and the parent 'Jiyou No. 1' showed an uplift type ( Figure 7 ), indicating non-affinity.

[0074] 2 ITS Identification of Mutant Strains of Auricularia heimuer

[0075] Figure 8 As shown in Table 3, the genetic distance between the mutant strains was calculated, indicating that the black fungus mutant strain JH-Y6 has genetic differences from the parent strain and is a new strain different from the parent strain.

[0076]

[0077] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of Auricularia auricular, characterized in that, The Auricularia heimuer strain is JH-Y6, and the preservation number is CCTCC No: M 20232280.

2. The Auricularia auricula-judae strain according to claim 1, characterized in that, The Auricularia heimuer JH-Y6 strain has the characteristics of high tyrosinase activity and high melanin yield.

3. The Auricularia auricula-judae strain of claim 1, characterized in that, The ITS sequence of the Auricularia heimuer JH-Y6 strain is shown in SEQ ID NO.

1.

4. The Auricularia auricula-judae strain of claim 1, characterized in that, The Auricularia heimuer JH-Y6 strain is obtained by screening the spores of the parent strain 'Ji You No. 1' through ARTP-ultraviolet combined mutagenesis.

5. The basidiospores, mycelium and fruiting bodies of the Auricularia heimuer JH-Y6 strain according to any one of claims 1-4.

6. The application of the Auricularia heimuer JH-Y6 strain according to any one of claims 1-4 and the basidiospores, mycelium or fruiting bodies according to claim 5 in Auricularia heimuer screening breeding and cultivation.

7. The application of the Auricularia heimuer JH-Y6 strain according to any one of claims 1-4 and the basidiospores, mycelium or fruiting bodies according to claim 5 in extracting tyrosinase.

8. The application of the Auricularia heimuer JH-Y6 strain according to any one of claims 1-4 and the basidiospores, mycelium or fruiting bodies according to claim 5 in extracting melanin.

9. The application of the Auricularia heimuer JH-Y6 strain according to any one of claims 1-4 and the basidiospores, mycelium or fruiting bodies according to claim 5 in preparing food.

10. The application of the Auricularia heimuer JH-Y6 strain according to any one of claims 1-4 and the basidiospores, mycelium or fruiting bodies according to claim 5 in preparing health products.

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